Cynthia Wetmore

Identification of cells in rat brain and peripheral tissues expressing mRNA for members of the nerve growth factor family

Cells expressing mRNA for hippocampus-derived neurotrophic factor (HDNF/NT-3) or brain-derived neurotrophic factor (BDNF) were identified by in situ hybridization. In the rat brain, HDNF mRNA was predominantly found in pyramidal neurons in CA1 and CA2 of the hippocampus. Lower levels of HDNF mRNA were found in granular neurons of the dentate gyrus and in neurons of the taenia tecta and induseum griseum. BDNF mRNA-expressing cells were more widely distributed in the rat brain, with high levels in neurons of CA2, CA3, and the hilar region of the dentate gyrus, in the external and internal pyramidal layers of the cerebral cortex, in the claustrum, and in one brainstem structure. Lower levels were seen in CA1 and in the granular layer of the hippocampus, in the taenia tecta, and in the mammillary complex. In peripheral tissues, HDNF mRNA was found in glomerular cells in the kidney, secretory cells in the male rat submandibular gland, and epithelial cells in secondary and tertiary follicles in the ovary. Cells expressing BDNF mRNA were found in the dorsal root ganglia, where neurons of various sizes were labeled.

Localization of brain-derived neurotrophic factor mRNA to neurons in the brain by in situ hybridization

Brain-derived neurotrophic factor (BDNF) which supports survival of and fiber outgrowth from sensory neurons and retinal ganglion cells has recently been cloned. It is closely related to nerve growth factor (NGF), which we had demonstrated earlier by in situ hybridization to be expressed in the brain by cholinergic target neurons. Using oligonucleotide probes we now describe aspects of the distribution of BDNF mRNA in the adult pig and rat brain. Similar to NGF, BDNF expression in vivo in the central nervous system appeared to be mostly confined to neurons. In the hippocampal formation, strongly labeled neurons were found in and around the pyramidal layer, as well as in the granular layer and the hilus of the dentate gyrus of both pig and rat. Other neurons in these areas were more weakly labeled or unlabeled. High BDNF expression was also shown in scattered neurons in cortex cerebri, in many neurons in claustrum and in certain other areas. These results demonstrate that the similarity in structure between NGF and BDNF is paralleled by a similarity in cellular expression, suggesting that BDNF, like NGF, may act via a direct neuron-to-neuron interaction. It is possible that some neurons, for example, in the hippocampal formation, express both NGF and BDNF. Finally, the localization of BDNF expression in cortical areas and in claustrum suggests that the target neurons for this factor extend beyond the sensory ganglion and retinal ganglion cells.

COMPARATIVE STUDY OF BRAIN-DERIVED NEUROTROPHIC FACTOR MESSENGER RNA AND PROTEIN AT THE CELLULAR LEVEL SUGGESTS MULTIPLE ROLES IN HIPPOCAMPUS, STRIATUM AND CORTEX

Brain-derived neurotrophic factor (BDNF) is important for the development and trophic support of several neuronal groups in the rat. In the present study, the distribution of BDNF messenger RNA was studied by in situ hybridization, and the cellular localization of BDNF protein was investigated with anti-peptide antibodies. Anatomical investigations were also made in animals with prolonged epileptic seizures which show an enhanced expression of BDNF messenger RNA. Major forebrain areas studied were the hippocampus, striatum and cortex. The messenger RNA coding for the putative high-affinity receptor, tyrosine kinase B, was also visualized using in situ hybridization with a probe specific for the full-length form. In the hippocampus, granule cells and pyramidal neurons expressed BDNF messenger RNA and BDNF-like immunoreactivity. Interneurons in dendritic layers did not show labelling with either method. Tyrosine kinase B messenger RNA was found within neurons in all these regions. In the medial septum-diagonal band, nucleus basalis and lateral hypothalamus, neurons with punctate cytoplasmic immunofluorescence were found, and neurons in the lateral septum were diffusely positive for BDNF. In striatum, positive labelling of medium-sized neurons was found with the antibody, whereas BDNF messenger RNA was only detectable during seizures. A laminar pattern of neuronal labelling for BDNF messenger RNA and protein was found in the neocortex. The analysis of the anatomical distribution of BDNF-producing cells suggests a number of possible cellular interactions. In the hippocampus, BDNF might act in an autocrine or paracrine manner for granule cells and pyramidal neurons, and, in addition, may serve as a signal from these principal cells to interneurons. BDNF could be a target-derived and a locally produced trophic factor for cholinergic neurons in the medial septum. The expression of BDNF in the striatum suggests that this factor could be a target-derived factor for dopaminergic neurons of substantia nigra and/or work as an autocrine/ paracrine factor within the striatum itself.

Regulation of brain-derived neurotrophic factor messenger RNA and protein at the cellular level in pentylenetetrazol-induced epileptic seizures

We have examined the effects of pentylenetetrazol-induced epileptic seizures on brain-derived neurotrophic factor messenger RNA and protein and on the messenger RNA of its receptor in the rat. Pentylenetrazol, which acts at the picrotoxin recognition site of the GABAA receptor, was injected intraperitoneally and induced seizures by decreasing the inhibitory GABAergic activity. The effects of a single acute convulsive dose (50 mg/kg) of pentylenetetrazol were analysed at different time points by in situ hybridization or immunohistochemistry. Kindling was induced by daily subconvulsive injections (30 mg/kg) of pentylenetetrazol. At different time points during the kindling process, the messenger RNAs of brain-derived neurotrophic factor and trkB and the protein levels of brain-derived neurotrophic factor were analysed. We showed that brain-derived neurotrophic factor messenger RNA dramatically increased in neurons of the granule cell layer, piriform cortex and amygdala 3 h but not 6 h after an acute high dose of pentylenetetrazol, while brain-derived neurotrophic factor-like immunoreactivity was decreased in the granule cell layer and neurons of the hilus. The trkB messenger RNA was similarly increased 3 h and 6 h after the injection and returned to control levels after 24 h. The first change during the kindling development was seen after the first severe seizure: brain-derived neurotrophic factor messenger RNA was markedly increased in the piriform cortex and amygdala but not in the hippocampus. In fully kindled rats, which had several severe seizures, brain-derived neurotrophic factor messenger RNA and trkB messenger RNA were unaffected 3 h and 24 h after the last pentylenetetrazol injection. However, brain-derived neurotrophic factor-like immunoreactivity was markedly increased in the hippocampal formation 3 h, 24 h and three days after the last pentylenetetrazol injection, and still increased after 10 days. These results suggest that brain-derived neurotrophic factor may be involved in protection mechanisms after damage during seizures and in sprouting responses. The piriform cortex/amygdala seems to be an area of origin for the kindling development.

Cellular hybridization for BDNF. trkB, and NGF mRNAs and BDNF-immunoreactivity in rat forebrain after pilocarpine-induced status epilepticus

The messenger RNAs (mRNAs) for the neurotrophins, brain-derived neurotrophic factor (BDNF), and nerve growth factor (NGF), are upregulated during epileptic seizure activity, as visualized by in situ hybridization techniques. Neurotrophins might be protective against excitotoxic cell stress, and the upregulation during seizures might provide such cell protection. In this study, a high dose of pilocarpine (300 mg/kg) was used to induce long-lasting, limbic motor status epilepticus and a selective pattern of brain damage. The regulation of BDNF, trkB, and NGF mRNA was studied by in situ hybridization at 1, 3, 6, and 24 h after induction of limbic motor status epilepticus. BDNF immunoreactivity was examined with an anti-peptide antibody and the neuropathological process studied in parallel. BDNF mRNA increased in hippocampus, neocortex, piriform cortex, striatum, and thalamus with a maximum at 3–6 h. Hybridization levels increased earlier in the resistant granule and CA1 cells as compared to the vulnerable CA3 neurons. BDNF immunoreactivity was elevated in dentate gyrus at 3–6 h. trKB mRNA increased in the entire hippocampus. NGF mRNA in hippocampus appeared in dentate gyrus at 3–6 h and declined in hilar neurons at 6–24 h. Cell damage was found in the CA3 area, entire basal cortex, and layers II/III of neocortex. Endogenous neurotrophins are upregulated during status epilepticus caused by pilocarpine, which is related to the coupling between neuronal excitation and trophic factor expression. This upregulation of neurotrophic factors may serve endogenous protective effects; however, the excessive levels of neuronal hyperexcitation resulting from pilocarpine seizures lead to cell damage which cannot be prevented by endogenous neurotrophins.

The nerve growth factor receptor gene is expressed in both neuronal and non-neuronal tissues in the human fetus

In situ hybridization was used to study expression of β‐nerve growth factor receptor (NGF‐R) mRNA in the early human fetus. In 8‐ to 12‐week old fetuses, high labelling was found over motoneurons along the entire length of the lateral motor column. High levels of NGF‐R mRNA were also seen over most developing nerve cell bodies in both the dorsomedial and ventrolateral part of the dorsal root ganglia. Lower, but clearly specific labelling was detected over a subpopulation of cells in Auerbachs plexus in the intestines. Evidence for a non‐neuronal expression of NGF‐R mRNA came from labelling over a subpopulation of cells in glomeruli of the kidney in a 12‐week old human embryo. Myoblasts in skeletal muscle anlagen were labelled as well as cells along peripheral nerve. The wide‐spread expression of NGF‐R mRNA in the human fetus suggests that the NGF‐R is important for development of a variety of different tissues of both neuronal and non‐neuronal origin.

Chapter 7 Grafts, growth factors and grafts that make growth factors

Publisher Summary This chapter presents an in vivo screening system based upon the intraocular grafting of fetal brain tissue, which can be used to monitor temporal and regional specificity of growth factor effects. It focuses on the most well-known nerve growth stimulating factor, nerve growth factor (NGF), and describes its presence in the central nervous system (CNS) as well as the fate of intraparenchymally injected NGF. The fate of NGF injected directly into brain parenchyma as monitored by immunohistochemical techniques is described in the chapter. Genetically engineered fibroblasts secreting large amounts of NGF that have been grafted to the CNS explain the way such cells can support surrounding cholinergic systems and rescue axotomized cholinergic neurons. The functions of NGF in the CNS might extend beyond the current set of cholinergic neurons. Thus, it is conceivable that NGF sensitivity is more widespread during development.

Initial studies of embryonic transplants of human hippocampus and cerebral cortex derived from schizophrenic women

Human fetal brain tissue was obtained from first-trimester elective abortions of two women who also had schizophrenia. Portions of the embryonic hippocampus or cerebral cortex were transplanted into the anterior eye chamber of immunologically compromised athymic nude rats. In this environment, embryonic brain tissue derived from normal women generally continues organotypic growth and development for many months. Although initial survival after transplantation was normal, the tissue derived from schizophrenic women manifested less robust growth. However, cells in the transplants showed typical neuronal differentiation, with development of different neuronal types, such as pyramidal cells, granule cells, and gamma-aminobutyric acid (GABA)-containing interneurons. Rhythmic electrical activity was also observed, indicative of some local synaptic organization. The presence of messenger RNA (mRNA) for brain-derived neuronotrophic factor (BDNF) was observed using in situ hybridization. The reason for the decreased rate of growth of these transplants remains unknown and the significance of the finding cannot be assessed from only two fetuses. However, these preliminary findings suggest that fetal transplants may be a useful model system for the detection of developmental pathogenic processes in the expression and transmission of schizophrenia.

High and low affinity NGF receptor mRNAs in human foetal spinal cord and ganglia.

The cellular localization of mRNAs encoding the low affinity NGF receptor (here referred to as LANR) and the putative high affinity receptor for NGF, trk, have been studied in the human foetal spinal and sympathetic ganglia, and spinal cord, using in situ hybridization. The receptor mRNAs were highly expressed in the spinal and sympathetic ganglia, with most but not all neurons expressing both LANR and trk mRNA. Spinal nerve rootlets distal to the spinal ganglia expressed LANR but not trk mRNA, confirming the presence of the low affinity receptor in developing Schwann cells. In the spinal cord, LANR mRNA was found throughout the medial and lateral motor columns while, trk mRNA was detected in scattered cells in the dorsal aspect of developing grey matter.

Brain-Derived Neurotrophic Factor and trkB Receptor mRNAs in Grafts of Cortex Cerebri

Trophic factors are expressed by neurons throughout several areas of the CNS. We studied the mRNA expression of a member of the neurotrophin family, brain-derived neurotrophic factor (BDNF), and of the two receptor transcripts, full-length trkB and truncated trkB in single intraocular cortex cerebri grafts and in double cortex cerebri grafts. All single as well as double intraocular cortex grafts grew well, reaching a maximal size 4 weeks postgrafting. BDNF mRNA was moderately expressed in neurons in all intraocular grafts and significantly increased compared to that in adult rat cortex. Truncated trkB mRNA was strongly expressed in neurons and glia, while full-length trkB mRNA was moderately expressed only in neurons in the intraocular cortical grafts. The expression of the two trkB transcripts in the grafted cortex did not differ from that of adult rat cortex. No difference in the expression of mRNAs for full-length or truncated trkB was found between single grafts grown for 4 or 8 weeks or between single and double grafts. Similarly, no difference in expression of BDNF mRNA in single grafts grown for 4 or 8 weeks was detected. However, BDNF mRNA levels were significantly lower in grafts which were placed in close contact with previously grafted cortex in the eye chamber. Moreover, contact with a second graft led to downregulation of BDNF mRNA in the first graft.(ABSTRACT TRUNCATED AT 250 WORDS)